1. Field of the Invention
The present invention relates to the role of inflammatory cells in the extension of tissue necrosis surrounding a thermally injured tissue. The invention also relates to the field of therapeutic agents and methods for the prevention and inhibition of tissue necrosis incident thermal injury in an animal.
2. Description of the Related Art
Thermal injuries result in a cascade of events which initially produces an area of irreversible tissue destruction surrounded by a marginal zone of injury with reduced blood flow. The marginal zone of injury is referred to as a "zone of stasis". This "zone of stasis" is recognized as being at high risk of progressive tissue destruction. Particularly, during the post-injury period, the zone of stasis is subject to a progressive extension of the area of tissue loss (necrosis) surrounding the initially tissue damaged site.
The progression of tissue loss is due in part to microvascular damage at and around the injured site. Three cellular events are primarily responsible for this observed microvascular damage. These include:
1) The adherence of neutrophils (polymorphonuclear neutrophils, PMNs) to capillary endothelial cells (EC), which induces the release of products toxic to host tissue; PA1 2) The aggregation of neutrophils, which causes clogging of capillaries and post-capillary venules; and PA1 3) The local activation of neutrophils to produce mediators of tissue damage (nitric oxide, etc.). PA1 PMN=polymorphonuclear neutrophil PA1 EC=endothelial cell PA1 CR3=complement receptor type 3 PA1 PMA=phorbol myristate acetate PA1 Hct=hematocrit PA1 BF=blood flow PA1 WBC=white blood cell PA1 CD11/CD18=a neutrophil membrane glycoprotein complex PA1 R 15.7=a monoclonal antibody to CD18 PA1 R 6.5=a monoclonal antibody to ICAM-1 PA1 ICAM-1=an endothelial cell intercellular adhesion molecule
The present invention provides for inhibiting the progressive tissue destruction typical of post-thermally injured patients through methods which inhibit each of these cellular events.
Leukocytes, particularly neutrophils, are central mediators of microvascular endothelial injury in many acute pathologic processes..sup.1-4 Leukocytes, particularly polymorphonuclear neutrophils (PMNs), monocytes and lymphocytes may be characterized as inflammatory cells, as cells of these cell types play some role in the inflammatory response..sup.1
Polymorphonuclear neutrophils are known to play a central role in inflammatory responses of many types. During inflammation, PMN-EC adherence results in the formation of a microenvironment between the PMN and the EC where PMN derived proteases (such as serine proteinase, elastase, collagenase and gelatinase), and toxic oxygen products produced by both the EC and PMN (such as superoxide anion, hydrogen peroxide and the hydroxyl radical), exist in high local concentrations..sup.1 These highly reactive substances, partially protected from inactivation by circulating plasma anti-proteases and free radical scavengers, then produce endothelial cell injury. This endothelial cell injury in turn results in intercellular gap formation, increased microvascular permeability, hemorrhage, edema and thrombosis.
PMN--PMN aggregation also occurs during inflammation, and further compromises the microvascular circulation by obstructing capillaries and post-capillary venules, thereby extending the zone of ischemia at the injury site. Because neutrophils (PMN's, particularly) are larger and less deformable than erythrocytes, they may plug small capillaries as perfusion pressure drops. Such a condition results in a mechanism of microvascular injury referred to as microvascular occlusion during ischemia-reperfusion..sup.2
PMNs have been shown to be activated during burn injury but their exact role as contributors to both the systemic and local microvascular occlusion seen following burn injury remains to be completely delineated. For example, Deitch et al. observed an increase in PMN activation when PMNs are exposed to burn blister fluid in vitro, including an increase in PMN complement receptor type 3 (CR3) expression..sup.6 Solem et al. have demonstrated an increase in the surface expression of a neutrophil membrane glycoprotein complex (CD18) on circulating PMNs following burn injury..sup.7
Several receptor-counter receptor pairs of ligands on the PMN and the EC have also been identified. One of the major receptor pairs is the neutrophil membrane glycoprotein complex, CD11-CD18 and its natural binding ligand, the intercellular adhesion complex (ICAM-1, CD54) on endothelial cells. The CD11/CD18 complex is a heterodimer composed of three distinct alpha chains CD11a, CD11b, and CD11c with a common beta chain, CD18. The CD11/CD18 complex, is present constitutively on the surface of normal neutrophils. The activity of the CD11/CD18 complex is upregulated in response to appropriate stimuli, such as to activated complement fragment 5a (c5a) or bacterial endotoxin.
ICAM-1 is a 76-97 kD glycoprotein (not a heterodimer) present on the endothelial cell (EC). Its expression by EC is upregulated by a number of inflammatory cytokines, including IL-1 and interferon..sup.24
It is known that neutrophil-endothelial cell adherence results from the specific interaction of the CD18 complex with ICAM-1. Monoclonal antibodies to portions of the CD11/CD18 complex have been found to functionally inhibit neutrophil aggregation and PMN-EC adherence in vitro and in vivo..sup.1,6-10 One such antibody, R 15.7, is a murine derived IgG2a which recognizes a functional epitope on CD18..sup.11 R 15.7 has been demonstrated both in vitro and in vivo to effectively block neutrophil adherence and emigration to a variety of stimuli, including LPS, phorbol myristate acetate (PMA), N-formylmethionyl-leucyl-phenylalanine (FMLP), complement fragment 5a (C5a), leukotriene B4 (LTB4), interleukin-1 (IL-1), and tumor necrosis factor (TNF).
Anti-CD18 antibodies have been shown to protect against inflammation by inhibiting PMN-EC adherence in reported isolated myocardial and intestinal ischemia-reperfusion injury studies..sup.12-13 Improved survival and reduced organ injury following hemorrhagic shock in rabbits and in non-human primates treated with anti-CD18 antibody have also been reported..sup.14
The interaction of PMN-EC has also been modulated through the use of antibodies directed against the endothelial cell, particularly ICAM-1 in modulating inflammation. One such antibody, R 6.5, (ATCC #HB9580) is a murine-derived IgG2a..sup.20 Administration of R 6.5 has been reported to reduce in vivo PMN migration in experimental rabbit models of airway inflammation. Such treatment reportedly was effective in preventing and treating acute renal rejection in nonhuman primates..sup.15-16 These antibodies have also been described as inhibiting intercellular adhesion of cells of granulocyte or macrophage lineage.
ICAM-1 expression is upregulated by cytokines. Therefore, it appears to be important at sites of inflammation therapy in giving specificity. However, so far, there have been no beneficial consequences reported in regard to vascular tissue or the containment of tissue necrosis upon treatment with various antibody preparations.
While progression of tissue necrosis continues to be a significant pathology attendant thermal injury in patients, no specific methods have yet been developed to treat/inhibit this type of secondary tissue loss. Moreover, the molecular mechanisms which play a part in this type of progressive tissue necrosis still have not been well defined.
Discovery of a method to contain the process of progressive tissue damage would provide for the development of improved methods for managing the burn or other thermally injured patient. Secondary tissue loss attendant burn injury contributes to the severity of the thermal injury, as well as to the overall disability, disfigurement and mortality risks observed in patients suffering from serious thermal injury. Therefore, methods for inhibiting and/or preventing tissue necrosis progression would also improve the ultimate prognosis of the thermally injured patient.